General Methods for Examination of Fungi.pdf

General Methods for Examination of Fungi Danielle Marie A. Allarey-Susa, RMT FUNGAL MEDIA AND CULTURE FUNGAL MEDIA AND CULTURE Identifying from culture Incubation is at 22°C-30°C If suspected for dimorphic fungus, cultures should also be incubated at 37°C Maintained for 4-6 weeks...

General Methods for Examination of Fungi Danielle Marie A. Allarey-Susa, RMT FUNGAL MEDIA AND CULTURE FUNGAL MEDIA AND CULTURE Identifying from culture Incubation is at 22°C-30°C If suspected for dimorphic fungus, cultures should also be incubated at 37°C Maintained for 4-6 weeks Record the ff: ✓Number of days required to see fruiting structures ✓Mold or yeast ✓Media used ✓Temperature ✓Morphology of colonies Colony Morphology 1. Texture 2. Growth Measurements 3. Center and Margin of Culture 4. Sulcation 5. Exudates 6. Reverse Colony 7. Any soluble pigments Colony Morphology 1. Texture 2. Growth Measurements 3. Center and Margin of Culture 4. Sulcation 5. Exudates 6. Reverse Colony 7. Any soluble pigments Colony Morphology 1. Texture 2. Growth Measurements 3. Center and Margin of Culture 4. Sulcation 5. Exudates 6. Reverse Colony 7. Any soluble pigments Colony Morphology 1. Texture 2. Growth Measurements 3. Center and Margin of Culture 4. Sulcation 5. Exudates 6. Reverse Colony 7. Any soluble pigments Colony Morphology 1. Texture 2. Growth Measurements 3. Center and Margin of Culture 4. Sulcation 5. Exudates 6. Reverse Colony 7. Any soluble pigments Colony Morphology 1. Texture 2. Growth Measurements 3. Center and Margin of Culture 4. Sulcation 5. Exudates 6. Reverse Colony 7. Any soluble pigments Fungal Culture Media Sabouraud Dextrose Agar (SDA) General medium Most commonly used 4% dextrose and an acidic pH Addition of inhibitors to make media selective ❖ Chloramphenicol ❖ Gentamicin ❖ Cycloheximide Fungal Culture Media Sabouraud Dextrose Agar (SDA) Modifications: Emmon’s Modification: 2% dextrose in Neutral pH Mycosel Agar: SDA + Chloramphenicol and Cycloheximide Fungal Culture Media Brain Heart Infusion (BHI) Agar Enrichment agar Recovery of Cryptococcus neoformans and dimorphic transitions of Sporothrix schenckii and Paracoccidioides brasilliensis Plates or tubes Modifications: BHI Broth + Penicillin = growth of Zygomycetes BHI + Gentamicin + Chloramphenicol = Cryptococcus neoformans from contaminated specimens Fungal Culture Media Inhibitory Mold Agar Enriched selective medium Contains inorganic salts, chloramphenicol and gentamicin Primary recovery of pathogenic fungi Sabouraud Dextrose + BHI (SABHI) General purpose medium Addition of blood increases isolation of dimorphic fungi Promotes conversion to the yeast stage Fungal Culture Media Dermatophyte Test Medium Enhance growth of dermatophytes in cutaneous specimens and inhibit other fungi and bacteria Cycloheximide, Chloramphenicol and Gentamicin Fungal Culture Media CHROMagar Candida Selective and differential for presumptive identification of genus Candida from primary plates Fungal Culture Media Cornmeal Tween 80 Agar Isolation of Candida albicans specifically for the growth of Chlamydospores Addition of trypan blue to provide contrasting background for observing morphologic features of yeast Christensen’s Urea Agar Composed of 2% Urea, Phenol Isolation and identification of Cryptococcus, Trichosporon and Rhodotorula spp. Fungal Culture Media Czapek’s Agar Differential identification of Aspergillus spp. Sodium nitrate, sucrose and yeast extract Niger Seed Agar/Birdseed Agar/Staib’s Medium Identification of Cryptococcus neoformans Potassium nitrate, peptone, meal extract, sulfanilic acid, N,N-dimethyl- 1naphthylamine Fungal Culture Media Potato Dextrose Agar Enhances pigmentation and sporulation of dermatophytes Potato extract, D-glucose Rice Medium Differentiation of Microsporum canis (yellow pigment) to Microsporum auduinii (rice grains turn brown) White rice extract, polysorbate 80 Fungal Culture Media Urea Agar Detection of Cryptococcus spp, differentiation of Trichophyton mentagrophytes from Trichophyton rubrum Peptone, dextrose, sodium chloride, monopotassium phosphate, urea and phenol red Positive result: Pink Cottonseed Agar Conversion of mold to yeast form of Blastomyces dermatitidis Indirect Microscopic Examination through Culture Septate versus sparsely septate hyphae Hyaline or phaeoid hyphae Fruiting structures Type size, shape and arrangement of conidia Microscopic Examination (from cultures) Microscopic characteristics that should be observed: Septate/Aseptate Hyaline/Phaeiod Fruiting Structures Types, size, shape and arrangement of conidia Methods LPCB Tease Preparation Scotch Tape/Adhesive/Cellophane Preparation Riddell/Microculture Technique Microscopic Examination (from cultures) LPCB (Lactophenol Cotton Blue) Tease Mount Teasing needles are used to remove a portion of the mycelium from the middle third of the colony. The mycelia are placed in a drop of lactophenol cotton blue (LPCB) on a slide and gently teased apart using the needles. The mycelia are then placed into a drop of LPCB on a slide, a cover slip is added and the slide is examined microscopically. Disadvantage: disruption of conidia during the teasing process Microscopic Examination (from cultures) Cellophane Tape/Scotch Tape Preparation gently touching a piece of clear tape, sticky side down, to the surface of the colony and then removing it. The tape is placed onto a drop of LPCB on a slide and examined. Advantage: retention of conidial arrangement Disadvantage: potential contamination of the colony, should be read within 30 minutes and then discarded Microscopic Examination (from cultures) Slide Culture Technique/Riddell Technique Useful for demonstrating the natural morphology of fungal structures DIRECT MICROSCOPIC EXAMINATION METHODS AND OTHER MISCELLENEOUS TESTS FOR FUNGAL IDENTIFICATION Direct Microscopic Examination Directly from clinical specimen Rapid report to the physician Provides clue to the genus of the organism Might provide evidence of infection despite negative cultures 10-20% Potassium Hydroxide (KOH) Test Most common routine test Nail scrapings, hair, skin scales, thin slices of tissue Presumptive test for fungal infections Upon collection of sample, 10-20% KOH is added to the slide, wait for 15-30 minutes, then look for yeast or hyphae. DMSO (Dimethyl sulfoxide)-rapid breakdown of cellular debris without the need for heating KOH with Calcofluor White Stain Uses a fluorescence dye In need of a fluorescence microscope to visualize formation of yeast and hyphae Superior to KOH test India Ink/Nigrosin Stain CSF sample is used to examine presence of Cryptococcus neoformans Yeast surrounded by a halo (capsule) against a black background. Miscellaneous Tests for the Identification of Molds Miscellaneous Tests for the Identification of Molds Hair Perforation Test Sterile 5- to 10-mm hair fragments are floated on sterile water supplemented with a few drops of sterile 10% yeast extract. Conidia or hyphae from the dermatophyte in question are inoculated onto the water surface. Hair shafts are removed and microscopically examined in LPCB at weekly intervals for up to 1 month Miscellaneous Tests for the Identification of Molds Urease Test Differentiate T. mentagrophytes from T. rubrum - 5-day urease test at room temperature Christensen urea agar Miscellaneous Tests for the Identification of Molds Thiamine Requirement Single most useful nutritional test for dermatophytes Tubes of media with and without thiamine are inoculated with a tiny, medium free portion of the colony and observed for growth after 10 to 14 days Miscellaneous Tests for the Identification of Molds Trichophyton Agars 7 different Trichophyton agars number 1 through 7 are used to determine the nutritional requirements of the Trichophyton spp. Growth on Rice Grains Microsporum canis and almost all other dermatophytes grow except Microsporum auduinii which does not grow but turns rice grains brown. Miscellaneous Tests for the Identification of Yeasts Miscellaneous Tests for the Identification of Yeasts Germ Tube Production Only Candida albicans and Candida dubliniensis are identified to be germ tube positive Use of Serum or Plasma (Fetal bovine serum, expired FFP) Brain Heart Infusion Broth, Trypticase Soy Broth, Nutrient Broth can be used as an alternative Incubated at 37°C and must be read not beyond 3 hours Miscellaneous Tests for the Identification of Yeasts Carbohydrate Assimilation Assimilation tests identify which carbohydrates a yeast can use aerobically as a sole carbon source. Yeast isolates are suspended in an agar basal medium, pipetted into the wells, and incubated at 30° C for 72 hours. As sugars are assimilated, the wells become turbid with growth. Miscellaneous Tests for the Identification of Yeasts Potassium Nitrate Assimilation Using modified Potassium Nitrate (KNO3) Agar accurate method to determine the ability of yeast to use nitrate as the sole source of nitrogen Positive test result turns the medium blue, if negative it turns yellow Cryptococcus albidus (positive control) Candida albicans (negative control) Miscellaneous Tests for the Identification of Yeasts Temperature Studies Cryptococcus spp Weak growth at 35°C, no growth at 42°C, optimal growth at 25°C Candida spp. Most can grow up to 45°C Candida dubliniensis cannot grow at 45°C Miscellaneous Tests for the Identification of Yeasts Urease Inoculated at room temperature for 48 hours Positive for Urease Cryptococcus and Rhodotorula Most strains of Trichosporon spp. Negative for Urease test Candida spp. Molecular Detection PCR Use the DNA of fungi as a confirmatory in identification Serological Test Use of blood samples Complement Fixation Histoplasma capsulatum Coccidioides immitis Blastomyces dermatitidis Agglutination Cryptococcus neoformans Other dimorphic fungi Points to Remember Yeasts typically reproduce by budding, whereas molds often reproduce by forming spores. Fungi can be isolated from almost any type of clinical specimen. Fungal identification is based on a variety of characteristics, including macroscopic appearance, microscopic appearance, ability to grow at various temperatures, and biochemical reactions. Antifungal therapy is frequently ineffective when diagnosis is delayed.